Although several molecular clones of human T-cell leukemia virus type I (HTLV-I) have been reported and shown to express viral mRNAs and proteins in transfected cells, their ability to produce infectious virus particles has not previously been demonstrated. It is possible that proviruses are altered during cloning, but it is more likely that the inability to prove infectivity results from the intrinsic difficulty in establishing infections with this virus. This may result from: (i) highly restricted expression which limits both detection and spread of the virus through the culture, (ii) poor infectivity of HTLV-I particles due to virion or envelope lability, and (iii) cytopathic effects of the virus on productively infected cells. We have established conditions to examine the infectivity, in vivo and in vitro, of proviruses cloned in this laboratory. The proviral clone, pCS-HTLV, was previously shown to direct the expression of viral mRNAs and proteins in transfected cells. We have now shown that transfected human 293 cells release virus particles that band at the appropriate density after sucrose gradient centrifugation. Growth medium from transfected cells was used for infections of several established T-cell lines and primary T-lymphocytes. Productive infection was demonstrated by reverse transcriptase-polymerase chain reaction analysis of host cell mRNA. Furthermore, neonatal Fischer F344 rats were inoculated with plasmid DNA encoding the HTLV-I provirus. At 4 months post-inoculation (PI) HTLV-I DNA was detected in peripheral blood mononuclear cells (PBMCs) and could be detected at 12 months PI. PBMCs isolated from inoculated animals could be induced to express virus. Thus, this cloned provirus is infectious.